摘要
目的研究针对Ki67基因的短发夹状RNA(shRNA)表达载体pSilencer-Ki67对人肾癌细胞生长的抑制作用。方法将pSilencer-Ki67转染人肾癌786-0细胞,24、48、72、120、144 h后分别采用逆转录-聚合酶链反应(RT-PCR)、Western blot技术检测Ki67 mRNA、蛋白表达,噻唑蓝(MTT)比色法检测细胞增殖,原位末端标记法检测细胞凋亡。结果pSilencer-Ki67转染后48 h 786-0细胞Ki67mRNA表达(34．1±1．7)%、蛋白表达(42．6±1．7)%降低最明显,与空质粒对照组[(97．7±0．9)%、(97．3±1．6)%]比较差异均有统计学意义(P＜0．01)。pSilencer-Ki67处理后48 h细胞增殖抑制率(76．7±3．2)%最高,72 h凋亡细胞阳性率(74．3±5．9)%最高,与空质粒对照组[(7．0±0．5)%、(8．4±1．1)%]比较差异有统计学意义(P＜0．01)。pSilencer-Ki67的这些作用可持续144 h。结论pSilencer-Ki67能有效、持续抑制人肾癌786-0细胞生长,有望成为肾癌基因治疗的有效工具。
Objective To evaluate inhibitory effects of the recombinant plasmid of small hairpin RNA （shRNA） against Ki67 gene, named pSilencer-Ki67, on the growth of human renal carcinoma 786- 0 cells. Methods The pSilencer-Ki67 was transfected into human renal carcinoma cell line 786-0 cells. At 24, 48, 72,120 and 144 h posttransfection, the effects of pSilencer-Ki67 on the Ki67 expression of human renal carcinoma 786-0 cells were detected by RT-PCR and Western blot. The proliferation of 786-0 cells was measured by MTT assay. The apoptosis of 786-0 cells was tested by TUNEL assay. Resulls RTPCR and Western blot analyses revealed that the Ki67 expression levels of 786-0 cells treated with pSi- lencer-Ki67 were reduced significandy. The proliferation of 786-0 cells treated with pSilencer-Ki67 was inhibited and the apoptosis increased. The knock down of Ki67 and inhibition of proliferation and induction of apoptosis mediated by pSilencer-Ki67 were maintained for 144 h and the maximal effect achieved at 48 h or 72 h posttransfection. Conclusion The expression plasmid of shRNA against Ki67 gene can inhibit the proliferation and induce apoptosis for a long period by blocking Ki67 expression of human renal carcinoma cells. The inhibition of Ki67 expression by pSilencer-Ki67 may be a rational approach in gene therapy for renal cancer.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第6期744-746,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(30570385)
卫生部科学研究基金(WKJ2005-2-026)
关键词
肾癌
短发夹状RNA
质粒
基因治疗
Kidney carcinoma
Small hairpin RNAs
Plasmid
Gene therapy
